Transcriptional networks that control the development of organisms are precise, highly coordinated, and complex. This proposal seeks to understand how the same transcription factor can specify multiple distinct cell fates during the development of an organism. Specifically, we are interested in the transcription factor Olig2, which can promote both a motoneuron and an oligodendrocyte cell fate. We hypothesize that Olig2 is able to perform its multiple functions through interactions with other DNA binding proteins causing it to bind different targets in different cellular contexts. Testing this hypothesis using current methods for the analysis of transcription factors is difficult because they cannot trace binding throughout a cell lineage, making it impossible to correlate DNA-binding events in progenitor cells to the final cell fates of their progeny. We propose to use a novel method, transposon Calling Cards, to record Olig2 binding during neural differentiation. The method entails fusing the transposase of a transposon to a transcription factor, thereby causing it to direct the insertion of transposon DNA into the genome near where it binds. The transposon becomes a Calling Card that permanently marks the transcription factor's visit to that place in the genome. By recovering these Calling Cards along with some of the genomic DNA that flanks them and then determining their DNA sequences, it is possible to map the genome-wide binding history of the transcription factor. We propose to apply the Calling Card method to understand how Olig2 carries out its distinct functions. Since many important transcription factors perform more than one function during development, the insights that we gain from this work should be broadly applicable. Our specific aims are 1) to trace Olig2 binding through neural stem cell differentiation to understand how it promotes two distinct cell fates, 2) to confirm that differentially bound target genes promote motoneuron or oligodendrocyte cell fates, 3) to analyze Olig2 and Ngn2 binding in living zebrafish using Calling Card technology. These aims are feasible: we have successfully implemented the Calling Card method in both yeast and mammalian cells, and our preliminary results demonstrate transcription factor directed insertion of Calling Cards in zebrafish. We are confident that the rewards of further developing this technology and applying it to understand the process of cell fate specification will be substantial